Differences in molecular mechanisms between human papillomavirus (HPV)-induced oral cavity and oropharyngeal squamous cell carcinomas (OC/OP SCCs) and those associated with tobacco use lead to different responses to therapy, with patients having HPV-positive tumors having a better prognosis than those with HPV-negative tumors. We and others have shown that site-specific and global differences in DNA methylation exist between HPV(+) and HPV(-) tumors. 5-hydroxymethylcytosine (5hmC), once thought of as simply a transient step in the demethylation process, is now known to be aberrant with functional consequences in multiple cancers, however it has not yet been studied in OC/OP SCCs. Given suggestive evidence that 5hmC may also play a role in oral and oropharyngeal tumors, we propose to study 5hmC, its interactions with DNA methylation, and how these translate to functional changes in gene expression in HPV(+) and HPV(-) OC/OP SCCs. The goal of Aim 1 of this proposal will be to determine the extent and form of 5hmC involvement in HPV(+) and HPV(-) tumors by performing whole-genome 5hmC deep sequencing on a current set of OC/OP SCC frozen tumors and matched controls. In Aim 2, we will integrate the 5hmC data with whole-genome bisulfite sequencing data (which does not distinguish between 5hmC and 5mC marks), transcriptomics data measured via RNA-seq, and copy number variation data on the same tumors to determine how DNA methylation (5mC) and 5hmC deregulation affect the transcriptional programming in OC/OP SCCs and their relationship with clinical outcome. To help accomplish our goals, we will develop a bioinformatics method and tool for concurrent analysis and interpretation of 5mC and 5hmC deep sequencing data.